Proposed here is research to develop a combination of mass spectrometry instrumentation and techniques plus chemical and biochemical methods that will facilitate identification and near complete amino acid sequence analysis of intact proteins or large protein fragments on a chromatographic time scale. This research will make it possible to characterize multiple post-translational modifications, particularly those that exist o the same protein molecule and together regulate its biological activity. This research is driven by five major innovations in my lab: development of (a) electron transfer dissociation (ETD) for fragmentation of intact proteins, (b) ETD and IIPT (ion-ion proton transfer) chemistry to obtain N- and C-terminal sequence information from intact proteins, (c) front end ETD (FETD) that facilitates a 10-50 fold increase in sensitivity for intact proteins, (d) micro-column enzyme reactors that generate 3-10 kDa proteins fragments and provide 96% sequence coverage for monoclonal antibodies and (e) methodology for enrichment of O-GlcNAcylated peptides by boronic acid chemistry in non- aqueous solvents. Going forward, we will implement new IIPT reagents to extend the usable mass range to m/z 4,000, employ a combination of IIPT and parallel ion parking strategies to concentrate multiple charge states observed in protein ESI into a single lower charge state for protein identification by CAD, develop parallel ion parking strategies to minimize second generation ETD reactions and thus allow us to read complete protein sequences from both the n- and c- termini of intact proteins, build a longer linear ion tra to increase the number of protein ions that can be stored for analysis, extend the micro-column enzyme reactor concept to other proteases and solvents and then apply all of this development to the analysis of proteins in outer membrane vesicles secreted by the antibiotic resistant, gram negative bacteria, Neisseria gonorrhoeae. The ultimate goal is to be able to identify bacterial, virulence-factor proteins on a chromatographic time scale. The above technology will also be employed to characterize (a) proteolytic cleavage events on histones that alter the epigenetic code, (b) O- GlcNAc sites on ribosomal proteins, mitochondrial proteins in the heart, and all known human kinases, and (c) protein binding partners for RSK1 (ribosomal protein S6 Kinase) that plays a major role in breast cancer.